Abstract
The last few years has seen an ever-increasing interest in the exploitation of microalgae as recombinant platforms for the synthesis of novel bioproducts. These could be biofuel molecules, speciality enzymes, nutraceuticals, or therapeutic proteins, such as antibodies, hormones, and vaccines. This exploitation requires the development of new genetic engineering technologies for those fast-growing, robust species suited for intensive commercial cultivation in bioreactor systems. In particular, there is a need for routine methods for the genetic manipulation of the chloroplast genome, for two reasons: firstly, the chloroplast genetic system is well-suited to the targeted insertion into the genome and high-level expression of foreign genes; secondly, the organelle is the site of numerous biosynthetic pathways and therefore represents the obvious “chassis,” on which to bolt new metabolic pathways that divert the carbon fixed by photosynthesis into novel hydrocarbons, pigments, etc. Stable transformation of the algal chloroplast was first demonstrated in 1988, using the model chlorophyte, Chlamydomonas reinhardtii. Since that time, tremendous advances have been made in the development of sophisticated tools for engineering this particular species, and efforts to transfer this technology to other commercially attractive species are starting to bear fruit. In this article, we review the current field of algal chloroplast transgenics and consider the prospects for the future.
Similar content being viewed by others
Abbreviations
- GOI:
-
gene-of-interest
- IPTG:
-
isopropyl-β-D-thiolgalactopyranoside
- UTR:
-
untranslated region
References
Bhattacharya, D., Yoon, H.S., and Hackett, J.D., Photosynthetic eukaryotes unite: endosymbiosis connects the dots, BioEssays, 2004, vol. 26, pp. 50–60.
Green, B.R., Chloroplast genomes of photosynthetic eukaryotes, Plant J., 2011, vol. 66, pp. 34–44.
Barkan, A., Expression of plastid genes: organelle-specific elaborations on a prokaryotic scaffold, Plant Physiol., 2011, vol. 155, pp. 1520–1532.
Wang, H.H., Yin, W.B., and Hu, Z.M., Advances in Chloroplast Engineering, J. Genet. Genom., 2009, vol. 7, pp. 387–398.
Wani, S.H., Haider, N., Kumar, H., and Singh, N.B., Plant plastid engineering, Curr. Genom., 2010, vol. 11, pp. 500–512.
Bowsher, C.G. and Tobin, A.K., Compartmentation of metabolism within mitochondria and plastids, J. Exp. Bot., 2001, vol. 52, pp. 513–527.
Purton, S., Tools and techniques for chloroplast transformation of Chlamydomonas, Adv. Exp. Med. Biol., 2007, vol. 616, pp. 34–45.
Radakovits, R., Jinkerson, R.E., Darzins, A., and Posewitz, M.C., Genetic engineering of algae for enhanced biofuel production, Euk. Cell, 2010, vol. 9, pp. 486–501.
Mayfield, S.P., Manuell, A.L., Chen, S., Wu, J., Tran, M., Siefker, D., Muto, M., and Marin-Navarro, J., Chlamydomonas reinhardtii chloroplasts as protein factories, Curr. Opin. Biotechnol., 2007, vol. 18, pp. 126–133.
Johanningmeier, U. and Fischer, D., Perspective for the use of genetic transformants in order to enhance the synthesis of the desired metabolites: engineering chloroplasts of microalgae for the production of bioactive compounds, Adv. Exp. Med. Biol., 2010, vol. 698, pp. 144–151.
Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R., Randolph-Anderson, B.L., Robertson, D., Klein, T.M., and Shark, K.B., Chloroplast transformation in Chlamydomonas with high velocity microprojectiles, Science, 1988, vol. 240, pp. 1534–1538.
Taylor, N.J. and Fauquet, C.M., Microparticle bombardment as a tool in plant science and agricultural biotechnology, DNA Cell Biol., 2002, vol. 21, pp. 963–977.
Blowers, A.D., Bogorad, L., Shark, K.B., and Sanford, J.C., Studies on Chlamydomonas chloroplast transformation: foreign DNA can be stably maintained in the chromosome, Plant Cell, 1989, vol. 1, pp. 123–132.
Goldschmidt-Clermont, M., Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of Chlamydomonas, Nucleic Acids Res., 1991, vol. 19, pp. 4083–4089.
Bateman, J.M. and Purton, S., Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker, Mol. Gen. Genet., 2000, vol. 263, pp. 404–410.
Rochaix, J.D., Chlamydomonas reinhardtii as the photosynthetic yeast, Annu. Rev. Genet., 1995, vol. 29, pp. 209–230.
Stern, D.B., The Chlamydomonas Sourcebook, New York: Academic, 2009, vol. 2.
Cardi, T., Lenzi, P., and Maliga, P., Chloroplasts as expression platforms for plant-produced vaccines, Expert Rev. Vaccines, 2010, vol. 9, pp. 893–911.
Scotti, N., Rigano, M.M., and Cardi, T., Production of foreign proteins using plastid transformation, Biotechnol. Adv., 2012, vol. 30, pp. 387–397.
Robertson, D.E., Jacobson, S.A., Morgan, F., Berry, D., Church, G.M., and Afeyan, N.B., A new dawn for industrial photosynthesis, Photosynth. Res., 2011, vol. 107, pp. 269–277.
Kindle, K.L., Richards, K.L., and Stern, D.B., Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii, Proc. Natl. Acad. Sci. USA, 1991, vol. 88, pp. 1721–1725.
Doetsch, N.A., Favreau, M.R., Kuscuoglu, N., Thompson, M.D., and Hallick, R.B., Chloroplast transformation in Euglena gracilis: splicing of a group III twintron transcribed from a transgenic PsbK operon, Curr. Genet., 2001, vol. 39, pp. 49–60.
Gutiérrez, C.L., Gimpel, J., Escobar, C., Marshall, S.H., and Henríquez, V., Chloroplast genetic tool for the green microalgae Haematococcus pluvialis (Chlorophyceae, Volvocales), J. Phycol., 2012, vol. 48, pp. 976–983.
Lapidot, M., Raveh, D., Sivan, A., Arad, S.M., and Shapira, M., Stable chloroplast transformation of the unicellular red alga Porphyridium species, Plant Physiol., 2002, vol. 129, pp. 7–12.
Remacle, C., Cline, S., Boutaffala, L., Gabilly, S., Larosa, V., Barbieri, M.R., Coosemans, N., and Hamel, P.P., The ARG9 gene encodes the plastid-resident N-acetyl ornithine aminotransferase in the green alga Chlamydomonas reinhardtii, Euk. Cell, 2009, vol. 8, pp. 1460–1463.
Franklin, S., Ngo, B., Efuet, E., and Mayfield, S.P., Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast, Plant J., 2002, vol. 30, pp. 733–744.
Sakamoto, W., Kindle, K.L., and Stern, D.B., In vivo analysis of Chlamydomonas chloroplast PetD gene expression using stable transformation of beta-glucuronidase translational fusions, Proc. Natl. Acad. Sci. USA, 1993, vol. 90, pp. 497–501.
Matsuo, T., Onai, K., Okamoto, K., Minagawa, J., and Ishiura, M., Real-time monitoring of chloroplast gene expression by a luciferase reporter: evidence for nuclear regulation of chloroplast circadian period, Mol. Cell. Biol., 2006, vol. 26, pp. 863–870.
Mayfield, S.P. and Schultz, J., Development of a luciferase reporter gene, LuxCt, for Chlamydomonas reinhardtii chloroplast, Plant J., 2004, vol. 37, pp. 449–458.
Michelet, L., Lefebvre-Legendre, L., Burr, S.E., Rochaix, J.-D., and Goldschmidt-Clermont, M., Enhanced chloroplast transgene expression in a nuclear mutant of Chlamydomonas, Plant Biotechnol. J., 2011, vol. 9, pp. 565–574.
Rasala, B.A., Muto, M., Sullivan, J., and Mayfield, S.P., Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5’ untranslated region optimization, Plant Biotechnol. J., 2011, vol. 9, pp. 674–683.
Fletcher, S.P., Muto, M., and Mayfield, S.P., Optimization of recombinant protein expression in the chloroplasts of green algae, Adv. Exp. Med. Biol., 2007, vol. 616, pp. 90–98.
Kato, K., Marui, T., Kasai, S., and Shinmyo, A., Artificial control of transgene expression in Chlamydomonas reinhardtii chloroplast using the Lac regulation system from Escherichia coli, J. BioSci. Bioeng., 2007, vol. 104, pp. 207–213.
Surzycki, R., Cournac, L., Peltier, G., and Rochaix, J.-D., Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas, Proc. Natl. Acad. Sci. USA, 2007, vol. 104, pp. 17 548–17 553.
Wu, S., Xu, L., Huang, R., and Wang, Q., Improved biohydrogen production with an expression of codonoptimized hemH and lba genes in the chloroplast of Chlamydomonas reinhardtii, Bioresour. Technol., 2011, vol. 102, pp. 2610–2616.
Ellis, T., Adie, T., and Baldwin, G.S., DNA assembly for synthetic biology: from parts to pathways and beyond, Integr. Biol. (Camb), 2011, vol. 3, pp. 109–118.
Dreesen, I.A.J., Charpin-El, HamriG., and Fussenegger, M., Heat-stable oral alga-based vaccine protects mice from Staphylococcus aureus infection, J. Biotechnol., 2010, vol. 145, pp. 273–280.
Yoon, S.-M., Kim, S.Y., Li, K.F., Yoon, B.H., Choe, S., and Kuo, M.M.-C., Transgenic microalgae expressing Escherichia coli AppA phytase as feed additive to reduce phytate excretion in the manure of young broiler chicks, Appl. Microbiol. Biotechnol., 2011, vol. 91, pp. 553–563.
Gregory, J.A., Li, F., Tomosada, L.M., Cox, C.J., Topol, A.B., Vinetz, J.M., and Mayfield, S., Algaeproduced Pfs25 elicits antibodies that inhibit malaria transmission, PLoS ONE, 2012, vol. 7: e37179.
Jones, C.S., Luong, T., Hannon, M., Tran, M., Gregory, J.A., Shen, Z., Briggs, S.P., and Mayfield, S.P., Heterologous expression of the C-terminal antigenic domain of the malaria vaccine candidate Pfs48/45 in the green algae Chlamydomonas reinhardtii, Appl. Microbiol. Biotechnol., 2012, May 18 [Epub ahead of print].
Specht, E., Miyake-Stoner, S., and Mayfield, S., Micro-algae come of age as a platform for recombinant protein production, Biotechnol. Lett., 2010, vol. 32, pp. 1373–1383.
Tran, M., Zhou, B., Pettersson, P.L., Gonzalez, M.J., and Mayfield, S.P., Synthesis and assembly of a full-length human monoclonal antibody in algal chloroplasts, Biotechnol. Bioeng., 2009, vol. 104, pp. 663–673.
Fukusaki, E.-I., Nishikawa, T., Kato, K., Shinmyo, A., Hemmi, H., Nishino, T., and Kobayaski, A., Introduction of the archaebacterial geranylgeranyl pyrophosphate synthase gene into Chlamydomonas reinhardtii chloroplast, J. BioSci. Bioeng., 2003, vol. 95, pp. 283–287.
Tan, C.-P., Zhao, F.-Q., Su, Z.-L., Liang, C.-W., and Qin, S., Expression of β-carotene hydroxylase gene (crtR-B) from the green alga Haematococcus pluvialis in chloroplasts of Chlamydomonas reinhardtii, J. Appl. Phycol., 2007, vol. 19, pp. 347–355.
Su, Z.-L., Qian, K.-X., Tan, C.-P., Meng, C.-X., and Qin, S., Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii, Acta Biochim. Biophys. Sinica (Shanghai), 2005, vol. 37, pp. 709–712.
Szaub, J.B., Genetic engineering of green microalgae for the production of biofuel and high value products, Ph.D. Dissertation, London: Univ. College London, 2012.
Materna, A.C., Sturm, S., Kroth, P.G., and Lavaud, J., First induced plastid genome mutations in an alga with secondary plastids: PsbA mutations in the diatom Phaeodactylum tricornutum (Bacillariophyceae) reveal consequences on the regulation of photosynthesis, J. Phycol., 2009, vol. 45, pp. 838–846.
Fischer, N., Stampacchia, O., Redding, K., and Rochaix, J.D., Selectable marker recycling in the chloroplast, Mol. Gen. Genet., 1996, vol. 251, pp. 373–380.
Adachi, T., Takase, H., and Tomizawa, K., Introduction of a 50 kbp DNA fragment into the plastid genome, BoiSci. Biotechnol. Biochem., 2007, vol. 71, pp. 2266–2273.
Author information
Authors and Affiliations
Corresponding author
Additional information
This text is published in original.
Rights and permissions
About this article
Cite this article
Purton, S., Szaub, J.B., Wannathong, T. et al. Genetic engineering of algal chloroplasts: Progress and prospects. Russ J Plant Physiol 60, 491–499 (2013). https://doi.org/10.1134/S1021443713040146
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S1021443713040146